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Abstract

A simple, accurate and sensitive spectroscopic method has been proposed for the assay of ciprofloxacin in tablet by least square treatment of fourier transform infrared spectrometric data obtained at the wavenumber corresponding to the carbonyl group centered at 1707 cm-1. The method involves the extraction of the active ingredient with methanol followed by phosphate buffer pH 6.0. The excipients in the commercial tablet preparation did not interfere with the assay. The specifity, linearity, detection limits, precision and accuracy of the calibration curve, drug extraction, infrared analysis and data manipulation were determined in order to validate the method. Moreover, the statistical results were compared with other methods for quantification of ciprofloxacin.

Keywords

Ciprofloxacin, FTIR, method validation

Introduction

Ciprofloxacin hydrochloride, 1-cyclopropyl-6- fluoro-
1,4-dihydro-4-oxo-7-(1-piperazinyl)-3- quinoline
carboxilic acid (CPX), is a broad spectrum
fluoroquinolone antibacterial agent used in the
treatment of various bacterial infections caused by
gram-positive and gram-negative microorganisms [1,2]
(fig. 1). CPX is official in IP, USP and BP and their monograph revealed that RP- HPLC method
(IP and USP) and non-aqueous titrimetric method
(BP) were described for its estimation [3-5]. Others
workers have reported methods ranging from
derivatization coupled with ion-pair complexation
reaction, capillary electrophoresis, high performance
liquid chromatography (HPLC), colorimetry to
spectrophotometric technique [5-8]. The literature
shows variety of methods to analyze raw CPX
and in pharmaceutical preparation. The recovery studies for non-aqueous titrimetric assay [9] using
tetra-N-butylammonium hydroxide as titrant and
pyridine as solvent; the visual titrimetric, pH metric,
conductometric and spectrophotometric [10] techniques
for CPX as bulk drug and in commercial formulations
were found satisfactory.

Fig. 1: Structural formula of Ciprofloxacin hydrochloride

Potentiometric titration avoids the interference of the
excipients since completion of reaction is detected
through slope change in graph between EMF (or pH)
vs volume of titrant. The work is based on the fast
complexation reaction between iron (III) and CPX
in sulfuric acid media by using silver amalgam
electrode [11]. The spectrofluorimetric assay [12] was
performed by using wavelengths of excitation and
emission at 290 and 450 nm, respectively and
Spectrophotometric determination following oxidation
with cerium sulphate at λmax 470 nm [13]. The capillary
zone electrophoresis (CZE) has been elaborated for
separation, identification and determination of CPX
and its impurities. The phosphate buffer (pH 6.0)
was supplemented with 0.075 M pentane-1-sulfonic
acid sodium salt. The validation results were found
applicable to the analysis of different medicinal
products containing CPX [14].

The HPLC method is used worldwide for quality
control of CPX. This method allows determination of
CPX in different pharmaceutical dosage forms in the
presence of its potential impurities and degradation
products [15,16].

However, to date, no analytical method has been
described in the literature that uses fourier transform
infrared spectroscopy for quantitative analysis of
CPX in pharmaceutical tablet. The aim of the present study is to investigate the validation and application
of analytical methods for quality control of 100 mg
CPX hydrochloride tablet formulations.

An accurately weighed quantity of CPX HCl was
dissolved in small quantity of phosphate buffer
pH 6.0 and the final volume was made up to
100 ml with methanol as solvent to get 500 μg/
ml concentration of standard stock solution. Various
working concentrations were made by further dilution
with same medium.

Ten tablets were taken for assay, finely powdered
and homogenized. An accurately weighed quantity
of powder was dissolved in 5 ml phosphate buffer
pH 6.0 followed by 20 ml of methanol. The solution
is maintained under stirring for 5 min and was
centrifuged in order to separate excipients. The
supernatant solution was further diluted with 25 ml
of methanol to get 150 μg/ml solutions. All the
determinations were conducted in triplicate taking
the absorbance of each solution at 1707 cm-1 wavenumber. The method validation was done by
evaluating selectivity and linearity, limit of detection
(LOD) and limit of quantitation (LOQ), accuracy,
precision as indicated in the ICH guidelines [17].

For the Selectivity and linearity, the primary stock
of CPX solution was diluted as appropriate with
methanol and phosphate buffer to obtain final
dilutions in three replicates. The calibration curve for
CPX was obtained by plotting the peak area (carbonyl
group centered at 1707 cm-1) versus concentration.

The LOD is the lowest concentration of an analyte
that the analytical process can reliably detect and
the LOQ refers to the smallest concentration or the mass which can be quantitatively analyzed with
reasonable reliability by a given procedure [18]. The
LOD and LOQ were calculated by instrumental and
statistical methods. For the instrumental method LOD
is determined as the lowest amount to detect and
LOQ is the lowest amount to quantify by the detector.
For statistical method LOD and LOQ determined
by statistical formula. LOD= 3.3 σ/Slope, LOQ=
10 σ/ Slope, where, σ is standard deviation.

Accuracy of an analytical method describes the
closeness of mean test results obtained by the method
to the true value (concentration) of the analyte. This
is sometimes termed as trueness [19]. The statistical
accuracy was determined by adding known amount
of CPX reference standard to the sample at the
beginning of the process. Amount of 1.0, 2.0 and
3.0 ml of CPX standard solution (500 μg/ml) and
a aliquot of 2 ml of this solution (150 μg/ml) was
transferred to 100 ml volumetric flask, respectively
B1, B2 and B3. The percentage recovery was also
performed for B1, B2, and B3 for three days in
triplicate. Precision is the closeness of individual
measures of an analyte when the procedure is applied
repeatedly. The % Relative standard deviation
(RSD) is indication of repeatability (precision) and
reproducibility of an experiment. %RSD of peak areas
of carbonyl group was calculated for B1, B2 and B3
solution.

The accuracy and precision of the assay, as well
linearity of the calibration curve, were determined.
Having established the quantitative relationships
between the parameters studied, and knowing the
predictive performance of their association model, a
linear simple regression by the least squares method
was applied. The statistical analysis was calculated
by ANOVA.

The linearity results were evaluated by linear
regression analysis based on the minimum square
method. The correlation coefficient value obtained
over the range of 2-20 μg/ml is r=0.998. The
result shows linear correlation between analytical
responses and drug concentration. The ability to
assess unequivocally the analyte in presence of
excipients was tested. The method is found to be
selective (fig. 2).

The LOD and LOQ is found 0.068 μg/ml and
0.450 μg/ml respectively. The %RSD values are indication of repeatability and reproducibility of an
experiment and the error and recovery values shows
Accuracy of analytical determination. The FTIR
analytical method showed good accuracy, evaluated
by the recovery test (Table 1). The mean recovery
percentages for products B1, B2 and B3 respectively
101.72, 98.34, 100.41, corroborated satisfactory
recovery.

Control samples

Amount of standard added

% Recovery

%RSD

% Mean Recovery

(500 µg/ml)

Day1

Day2

Day3

B1

1 ml

103.2

97.8

104.1

3.37

100.72±3.42

B2

2 ml

101.2

98.3

95.55

2.89

98.34±2.83

B3

3 ml

103.2

98.9

99.14

2.42

100.41±2.43

N=3

Table 1: Accuracy Data

The precision was determined by means of a oneway
ANOVA including 3 replicates carried out
on three successive days The percentage relative
standard deviations for repeatability and intermediate
precision were 1.16 and 0.73. These experimental
results confirmed good precision of the method when
performed on the same or different days by different
analysts (Table 2).

Control samples

Amount of standardadded (500µg/ml)

Intermidiate precision (mean value in µg/ml)

%RSD

Repeatability(mean valueµg/ml)

%RSD

Day1

Day2

Day3

B1

1 ml

753

758

722

1.29

752

1.91

B2

2 ml

1245

1231

1242

0.59

1233

0.99

B3

3 ml

1715

1722

1726

0.32

1728

0.59

N=3

Table 2: Precision Data

The robustness test examines the experimental
conditions for the method, and the potentially
responsible factors such as experimental and
environmental conditions, to be taken into account
during method development. To demonstrate the
robustness of the method the following conditions
were changed i.e. cuvette (CaF2 and KBr) and
temperature. For this sample solution B2 was used in
triplicate. For FTIR analytical methods, the low RSD
values demonstrated that the analysis factors (cuvette
and room temperature) did not have a significant
effect on analytical responses (Table 3).

Conditions

Average concentration

%RSD

Cuvette 1(CaF2)

1240

0.91

Cuvette 2 (KBr)

1215

1.23

Air conditioner turned off

1232

1.31

n=3

Table 3: Robustness Data

Table 4 shows labeled amount of active ingredient
and the percentage recovery values obtained for
CPX pharmaceuticals. The recovery values are
with in acceptable limits as recommended by
pharmacopeias (90-110% in BP, USP). Table 5 compares the linearity, accuracy, precision and recovery of present method with other techniques
reported in literatures [20,21]. The quantification of
CPX through FTIR method presents lower precision
than other methods reported in Table 5. The low
precision of FTIR, UV/Vis method might be attributed
to the extraction of the active ingredient from the
pharmaceutical that is required in order to perform
assay. The recovery values found were similar for all
methods.

All validation parameters were found to be highly
satisfactory, indicating linearity, selectivity, precision,
accuracy, robustness and adequate detection and
quantification limit. The method is therefore shown
to be suitable for evaluation of CPX hydrochloride
pharmaceutical tablet and for routine use in quality
control laboratories.

This technique extends the use of a standard IR
spectrophotometer, typically used for identification
purpose. The present method opens the possibility
of applying IR spectroscopy to quantify other active
ingredient than CPX.

Aksoy B, Kucukguzel I, Rollas S. Development and validation of a stability-indicating HPLC method for determination of ciprofloxacin hydrochloride and its related compounds in film-coated tablets. Chromatographia 2007;66:57-63.